RESEARCH ARTICLE

Summary. Microbial mat ecosystems are characterized by both seasonal anddiel fluctuations in several physicochemical variables, so that resident microorgan-isms must frequently adapt to the changing conditions of their environment. It hasbeen pointed out that, under stress conditions, bacterial cells with higher contentsof poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA con-tent. In the present study, PHA-producing strains from Ebro Delta microbial matswere selected using the Nile red dying technique and the relative accumulation ofPHA was monitored during further laboratory cultivation. The number of het-erotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/gmicrobial mat. Of these, 100 randomly chosen colonies were replicated on mineralsalt agar limited in nitrogen, and Nile red was added to the medium to detect PHA.Orange fluorescence, produced upon binding of the dye to polymer granules in thecell, was detected in approximately 10% of the replicated heterotrophic isolates.The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans werecompared with those of several of the environmental isolates spectrofluorometry.PHA accumulation, measured as relative fluorescence intensity, resulted in asteady-state concentration after 48 h of incubation in all strains assayed. At 72 h,the maximum fluorescence intensity of each strain incubated with glucose andfructose was usually similar. MAT-28 strain accumulated more PHA than the otherisolates. The results show that data obtained from environmental isolates can high-ly improve studies based on modeling-simulation programs, and that microbialmats constitute an excellent source for the isolation of PHA-producing strains withindustrial applications. [Int Microbiol 2006; 9(2):95-102]

Key words: Pseudomonas putida · P. oleovorans · poly-hydroxyalkanoates ·Ebro Delta microbial mats · Nile red · spectrofluorometry · screening

Rapid spectrofluorometricscreening of poly-hydroxyalka-noate-producing bacteria frommicrobial mats

Introduction

Microbial mats are stratified microbial ecosystems character-ized by cyclic seasonal fluctuations of flooding and desicca-tion, and by diel fluctuations in the concentrations of oxygen,sulfide, and other chemical nutrients. Microorganisms rapidly

respond to changes in various physicochemical gradients,and locate themselves according to the most favorable envi-ronmental conditions. This behavior is likely to govern thevertical species stratification that results from the activemigration of motile cells in response to the shifting gradientsof electron donors and/or acceptors observed within micro-bial mats [4,6,8,22]. Prokaryotes have evolved numerous

INTERNATIONAL MICROBIOLOGY (2006) 9:95-102www.im.microbios.org

Received 15 December 2005Accepted 20 February 2006

*Corresponding author:M. BerlangaDpt. of Microbiology and ParasitologyFaculty of PharmacyUniversity of BarcelonaAv. Joan XXIII, s/n08028 Barcelona, SpainTel. +34-934024497. Fax +34-934024498E-mail: mberlanga@ub.edu

Mercedes Berlanga1*M. Teresa Montero2

Jordi Hernández-Borrell2

Ricardo Guerrero3

1Department of Microbiologyand Parasitology, Faculty ofPharmacy, University ofBarcelona, Spain2Physical Chemistry Laboratory V,Faculty of Pharmacy, Universityof Barcelona, Spain3Department of Microbiology,Faculty of Biology, Universityof Barcelona, Spain

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mechanisms of resistance to stress conditions. For example,many microorganisms have an inherent ability to form restingstages (e.g., cysts and spores), which allows them to survive indesiccated environments [16]; others, such as the spirocheteSpirosymplokos deltaiberi, become swollen and form refractileresistant bodies on exposure to air [17]. Alternatively, otherbacteria exhibit a metabolic versatility in order to cope withfluctuations in the chemical conditions of their environment.

The accumulation of intracellular storage polymers isanother bacterial strategy that increases survival in a chang-ing environment [19]. Poly-hydroxyalkanoates (PHA) serveas an endogenous source of carbon and energy during starva-tion [13]. In Ebro Delta mats, anoxygenic photosynthesisrepresents about 26% of total organic carbon production [18]and PHA distribution may contribute about 2% of the organ-ic carbon of the photosynthetic layers [25]. The content ofPHA in the microbial mat usually increases over the day,reaching its maximum at 18:00 h, and decreases overnight,with a minimum at 6:00 h [20]. Thus, dusk and dawn areperiods of adaptation or accommodation to new physico-chemical environmental conditions within microbial mats.Consequently, the necessity for organisms to adapt toextreme environmental fluctuations by alternating betweenthe production and utilization of storage compounds mayexplain the diel pattern of in situ PHA accumulation observedin such environments [20]. More detailed, in situ analysis ofPHA and their repeating-unit composition in stratified photo-synthetic microbial mats from the Ebro Delta showed thehigher prevalence of hydroxyvalerate (HV) repeating unitsover hydroxybutyrate (HB) repeating units at approximately1 HB:2 HV [25].

PHA are members of a family of polyesters that includesa wide range of different D-hydroxyalkanoids acids charac-terized by the general chemical structure:

Since poly-β-hydroxybutyrate (PHB), one of the most abun-dant PHA, was first described in Bacillus megaterium byLemoigne in 1926, several studies have demonstrated theproduction of PHA by a wide variety of prokaryotes and alsoby several plants and animals. However, only prokaryotesaccumulate high-molecular-weight PHA in cytoplasmicgranules [9,27]. The granules are coated with a monolayer ofphospholipids and proteins. These granule-associated pro-teins play a major role in the synthesis and degradation ofPHA and in granule formation [23]. In Bacteria, PHA consti-

tute a major carbon and energy storage material, which isaccumulates when a carbon source is provided in excess andanother nutrient (such as nitrogen, sulfur, phosphate, iron,magnesium, potassium, or oxygen) is limiting. The polymer-ization of soluble intermediates into insoluble moleculesdoes not change the osmotic state of the cell, thereby avoid-ing leakage of these nutrient-rich compounds out of the cell.In addition, PHA-producing bacteria have the advantage ofnutrient storage at a relatively low maintenance cost and witha secured return of energy [15].

The analytical method first used to determine PHB accu-mulation in bacteria was based on the degradation of PHBwith sulfuric acid to crotonic acid, and measurement of thereaction at 235 nm by spectroscopy [14]. Later, other tech-niques were developed to measure PHA: gas chromatography[3], mass spectrometry [2], Fourier-transform infrared spec-troscopy [10], staining with the lipid coloring agent Nile redfollowed by spectrofluorometry [5,7], and flow cytometry[30]. Of these methods, Nile red fluorescence offers an easy,rapid screening technique that can be used to isolate potentialPHA-producing strains from environmental samples. In thepresent study, we used Nile red to select such strains from EbroDelta microbial mats and to monitor the relative amounts ofproduct (measured as relative fluorescence intensity) thataccumulated during laboratory cultivation.

Material and methods

Collection microorganisms. Two collection strains were used ascontrols in the screening of PHA-producing strains from Ebro Delta micro-bial mats and in monitoring the accumulation of PHA by spectrofluorome-try: Pseudomonas putida CECT 324 and P. oleovorans CECT 4079 as PHA-producing bacteria; and Escherichia coli ATCC 10536 as a non-PHA-produc-ing bacterium.

Isolation of PHA-accumulating heterotrophic bacteriafrom microbial mats. One gram of mat sample from Ebro Deltamicrobial mats was homogenized in 10 ml of Ringer ¼. A dilution series wasmade in Ringer ¼ to obtain 30–300 colony-forming units (cfu) per plate.Aliquots of 0.1 ml were plated onto Luria-Bertani (LB) agar (Scharlau,Barcelona, Spain) containing 1.0 % NaCl and on trypticase soy agar (TSA)(Scharlau). The plates were incubated for 24–48 h at 30ºC. Colonies thatdeveloped on agar were differentiated by color, elevation, form, and edgeappearance. Isolates of axenic colonies were randomly picked and culturedin solid mineral salt medium (MSM) containing (in g/l): agar 15, Na2HPO4 ·7H2O 6.7, NaCl 10, KH2PO4 1.5, NH4Cl 0.1, MgSO4·7H2O 0.2, CaCl2 0.01,ferrous ammonium citrate 0.06, and 1 ml trace elements [24]. MSM was sup-plemented with 5 g glucose/l and 0.5 mg Nile red (Sigma, St. Louis, MO,USA) (dissolved in dimethylsulfoxide)/ml [29]. Petri dishes were incubatedfor 4–5 days at 30ºC. The amount of orange fluorescence observed in PHA-positive colonies, particularly those of P. putida, was much higher than thebarely visible fluorescence of PHA-negative colonies, such as E. coli. Asample of each of the PHA+ isolates was smeared on a glass slide, heat-fixed,and stained with Nile blue A [21] to detect the presence of intracellular PHA

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granules. Briefly, the isolates were stained with a 1% aqueous solution ofNile blue A for 10 min, washed with water and 8% aqueous acetic acid for1 min to remove excess stain, dried with filter paper, and covered with aglass cover slip (to protect the stained cells from immersion oil). The prepa-ration was examined using an Olympus BX-40 epifluorescence microscopeand an excitation wavelength of approximately 460 nm. PHA granulesstained with Nile blue A fluoresced bright orange, with individual granulesoften visible within a cell.

General biochemical and physiological characteristics ofPHA-accumulating strains isolated from Ebro Deltamicrobial mats. PHA+ isolates were studied using several general bio-chemical and physiological probes, such as Gram stain, pigmentation onTSA, and temperature, pH, and salinity ranges. Oxidase and growth and fer-mentation on glucose, fructose, lactose, xylitol, arabinose, cellobiose, tre-halose, melibiose, rhamnose, galactose, xylose, sucrose, mannose were alsoanalyzed (Table 1S, ONLINE).

Spectrofluorometric monitoring of PHA accumulation.PHA was monitored spectrofluorometrically with Nile red as a fluorochromefollowing a modification of the procedure of Delegau et al. [5]. The microor-ganisms used in the assay were: E. coli, P. oleovorans, P. putida, and fivePHA+ isolates (MAT-07, MAT-13, MAT-16, MAT-17 and MAT-28) obtainedfrom the Ebro Delta microbial mats. These strains were grown overnight inTSB broth (3% NaCl) at 30ºC. Aliquots (1/100) from each culture were trans-ferred into 100-ml flasks containing nitrogen-limited MSM, glucose (5 g/l),and 0.5 μg Nile red dye (dissolved in dimethylsulfoxide)/ml. Liquid cultureswere incubated in an orbital shaker (100 rpm) at 30ºC for several days. At24, 48 and 72 h, a 1-ml sample was removed and then centrifuged in a micro-centrifuge at 10,000 rpm at room temperature. Pellets were washed in 1 mlof PBS (pH 7.0), suspended in 1 ml of 0.1 M glycine-HCl (pH 3.0), andincubated at room temperature in the dark for at least 2 h. The relativeamount of PHA within the cells, as indicated by the intensity of Nile-redorange fluorescence, was measured using an SLM Aminco 8100 spectroflu-orometer. The fluorescence excitation and emission wavelengths of thestained cells in 0.1 M glycine-HCl (pH 3) were 543 nm and 598 nm, respec-tively. Slits of excitation and emission were set to 10 nm at 900 V.

PHA accumulation after 72 h of incubation with glucose and fructosewas compared between the control strains and the isolates. The conditionsused in this assay were as explained above.

Transmission electron microscopy. One-milliliter aliquots of 48and 72 h cultures of MAT-28 in MSM with glucose were collected for TEManalysis. They were centrifuged in a microcentrifuge at 10,000 rpm at roomtemperature. Pellets were washed in 1 ml of PBS (pH 7.0), and fixed with2% of glutaraldehyde-PBS and then stained with osmium tetroxide anduranyl acetate. Samples were viewed in a Leica transmission electron micro-scope.

Results

Several different heterotrophic bacteria were isolated frommicrobial mats. Approximately 2.3 × 107 cfu/g mat wereobtained in TSA and ca. 104 cfu/g mat in LB agar with 1%salt. One hundred randomly chosen colonies were replicatedon agar/nitrogen-limited MSM containing glucose (5 g/l) andNile red. The dye produces orange fluorescence on binding topolymer granules in the cell [29]. Approximately 10% of

replicated heterotrophic isolates showed orange fluores-cence, while Nile blue A staining confirmed the presence ofintracellular lipidic granules (Fig. 1). The colonies exhibiteda diverse range of morphologies, although there were onlyminimal differences in the results of the biochemical analysis(Table1 and Table 1S ONLINE), which may reflect the fact thatthese microorganisms shared the same habitat. Most of theisolated strains were gram-negative and motile, able to growfrom pH 6 to pH 9, from 4 to 37ºC, and on 0.5–7% NaCl. Theisolates were oxidase-positive and showed no pigmentationon TSA. All of the isolates utilized the substrates assayed,some of them fermentatively (see Table 1S ONLINE).

Five of the PHA+ isolates, MAT-07, MAT-13, MAT-16,MAT-17 and MAT-28, were randomly selected for furtherstudy. The relative fluorescence intensity of strains cultured inMSM-glucose medium was tested in three conditions: (i) aftermore than 2 h of incubation in PBS buffer-Nile red (at room

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Fig. 1. (A) Fluorescent Nile red staining of strains from Ebro Delta micro-bial mats. The amount of orange fluorescence in poly-hydroxyalkanoate(PHA)-positive colonies was much higher than that of PHA-negativecolonies. (B) An isolate from Ebro Delta microbial mats observed by opticalmicroscopy. (C) The same microorganisms, stained with Nile blue dye,observed by epifluorescesce microscopy. PHA granules fluoresce brightorange within the cells. (Photograhs made by A. Navarrete.)

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temperature, in the dark); (ii) after more than 2 h of incuba-tion in 0.1 M glycine-HCl-Nile red (at room temperature, inthe dark); and (iii) immediate sonication in 0.1 M glycine-HCl-Nile red. The relative fluorescence intensity was lower

in cells stained with PBS-Nile red (condition i) than in thosestained with glycine-HCl-Nile red (condition ii). However,the relative fluorescence intensity in bacteria incubacted inglycine-HCl-Nile red (condition ii), was similar to that in

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Fig. 2. Transmision electron micrographs of strain MAT-28 grown on MSM medium with glucose for 48 h, and incubated for2 h in glycine-HCl-Nile red (A,B) and PBS-Nile red (C,D).

Table 1. Distinctive physiological characteristics of several PHA-producing strains isolated from EbroDelta microbial mats (see Table 1S ONLINE)

MAT-07 MAT-13 MAT-16 MAT-17 MAT-28

Gram stainRound, slimy coloniesOxidaseGrowth at 45ºCGrowth at 7% NaClArabinose fermentationa

Melibiose fermentationa

Galactose fermentationa

Xylose fermentationa

Mannose fermentationa

PHA productionb

Depolymerase (hydrolysis of PHA)

–++–––––++++

–++––++++++–

–+++–+++++++

–++–––+++–

+++

–++–+–+++–

++++

aFermentantion after 7 days of incubation at 30ºC.bProduction of PHA after growth in MSM-glucose at 30ºC, at 48–72 h.

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cells immediately sonicated (condition iii). This may be aconsequence of the disruption of the cell envelope when theywere incubated with glycine-HCl-Nile red (Fig. 2).

PHA accumulation, measured as relative fluorescenceintensity, was determined spectrofluorometrically in cellsgrown under the same conditions as stated above. The kineticsof PHA accumulation in P. oleovorans and E. coli were com-pared with those of four of the environmental isolates (Fig. 3).The experiments were done three times independently, and thevariation coefficient of each data point was less than 22%. Thelipid concentration in non-PHA-producing E. coli was lowerthan the level that could be detected spectrofluorometrically. Inall PHA-producing strains, accumulation of the polymer led toa steady-state concentration after approximately 48 h. Whengrown on glucose and fructose, the steady-state concentrationof PHA accumulated at 72 h, measured as the relative intensi-ty of Nile red fluorescence, was similar in all strains assayed,except for MAT-07 and MAT-13, which accumulated morePHA when grown on fructose. Of all the isolates, MAT-28accumulated the most PHA (Fig. 4).

Discussion

Among the different organisms that make up most of the matbiomass, there are heterotrophic microorganisms (e.g., fer-mentative bacteria, sulfur oxidizers) that do not form stable

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Fig. 3. (A) PHA accumulation in Pseudomonas oleovorans and the mat iso-lates MAT-07, MAT-13, MAT-16 and MAT-28 as measured spectrofluorome-trically. The final concentration of Nile red used in the assay was 0.5 μg/ml.The data shown are the average of the results of three independent experi-ments. In each case, the coefficient of variation (i.e., the standard devia-tion/mean × 100) was less than 22%. (B) Transmision electron micrographof MAT-28 growing on MSM medium with glucose at 48 h. (C) Transmisionelectron micrograph of MAT-28 at 72 h.

Fig. 4. PHA accumulation in Pseudomonas putida, P. oleovorans, and themat isolates MAT-07, MAT-13, MAT-16, MAT-17 and MAT-28 as measuredspectrofluorometrically at 72 h of incubation with glucose (white bars) orfructose (gray bars). The final concentration of Nile red used in the assaywas 0.5 μg/ml. The data shown are the average of the results of three inde-pendent experiments. In each case, the coefficient of variation (i.e., the stan-dard deviation/mean × 100) was less than 17%.

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layers; instead, their position in the mat depends on the avail-ability of organic matter and on other environmental vari-ables. Aerobic heterotrophic bacteria inhabiting microbialmats may play an important role in carbon cycling, yet infor-mation about their identity is scarce. Molecular phylogeneticstudies are currently underway to examine the microbialdiversity in naturally occurring mats. Preliminary findings of16S rRNA sequences in Guerrero Negro microbial matsshowed that heterotrophs were abundant, including α-, γ-, δ-,and ε-proteobacteria, firmicutes, spirochetes, and representa-tives of the Cytophaga/Flexibacter/Bacteroides (CFB)group. The variability in numbers of different kinds of organ-isms at different times and at different depths in the mats isextensive, reflecting the enormous complexity within boththe mats and individual strata [28]. Jonkers and Abed [12]were able to phylogenetically identify the apparent dominantpopulations of aerobic heterotrophs in a hypersaline micro-bial mat from Eilat by combining cultivation and moleculartechniques. The isolated populations were related to the gen-era Rhodobacter and Roseobacter (α-proteobacteria) and toMarinobacter and Halomonas (γ-proteobacteria).

We have used a cultivation step to obtain heterotrophsfrom Ebro Delta microbial mats, with the aim of isolatingpotential PHA-producing strains. The lipophilic dye Nile redcan be directly added to the medium to stain colonies thatstore PHA, and at concentrations that do not inhibit cellgrowth. Nile red staining thus allows both the detection ofPHA-producing strain from environmental samples andquantification of the polymer in liquid culture, resulting in asignificant reduction of analysis time and manpower.However, Nile red cannot be used to identify the monomercomposition of the accumulated PHA [7]. The fluorescenceintensity of stained cells depends on the PHA concentration.Bacteria that do not produce PHA are only lightly fluorescentbecause they contain almost no lipids [26] (see Figs. 1 and 2).In the PHA-producing strains isolated from Ebro Deltamicrobial mats, the kinetics of PHA accumulation were sim-ilar to those of P. putida, assayed under the same conditions.

In bacteria, PHA accumulation probably serves toincrease survival and stress tolerance in changing environ-ments and competitive settings, i.e., when carbon and energysources are limited, such as occurs in stratified microbialmats. Bacterial cells containing higher amounts of PHA, andthus larger nutrient stores, may survive longer than thosewith a lower PHA content. Alternatively, the polymer mayprotect against additional adverse factors [1,11]. Microbialmats (a complex biofilm) have a highly structured spatial dis-tribution of biotic and abiotic variables. This distribution

plays a significant role in determining the composition andactivity of the microbial populations that form the mat. Ourresults could be applied to modeling studies aimed at under-standing the intricate relationship between the structure andthe activity of the microbial community, as they allow thespatial distribution of the different populations in the matmatrix to be monitored according to substrate availability[8,18,31]. We propose that microbial mats constitute a poten-tial source for the isolation of new PHA-producing strains,although culture conditions must be optimized to obtainpolymer concentrations that are high enough to be industrial-ly and commercially exploited.

Acknowledgements. This work was supported by grant numberBOS2003-02944 and CGL2005-04990/BOS, from the Spanish Ministry ofScience and Technology; and by a DivMicCat grant from the Institute forCatalan Studies. We thank Carmen López of Scientific-TechnologicalServices of the University of Barcelona for TEM samples manipulation.

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Cribado espectrofluorométrico rápido debacterias productoras de polihidroxialcanoatosen tapetes microbianos

Resumen. El ecosistema de los tapetes microbianos se caracteriza porfluctuaciones diarias y estacionales en diversas variables fisicoquímicas, detal manera que los microorganismos residentes deben adaptarse frecuente-mente a las condiciones cambiantes de su ambiente. Se ha destacado que, encondiciones de estrés, las células bacterianas con elevado contenido en poli-hidroxialcanoatos (PHA) pueden sobrevivir más tiempo que las de bajo con-tenido. En este estudio, se utilizó la técnica del colorante rojo Nilo para sele-cionar las cepas productoras de PHA de los tapetes microbianos del Delta delEbro, y para monitorizar la acumulación relativa de PHA durante el cultivoen el laboratorio. El número de aislados heterotrofos en TSA fue de aproxi-madamente 107 unidades formadoras de colonias/g tapete microbiano. Deéstas, se replicaron 100 colonias elegidas al azar cultivadas en agar mineralsalino limitado en nitrógeno, al que se le añadió el colorante rojo Nilo parala detección de PHA. La fluorescencia naranja, que se produce al unirse elrojo Nilo a los gránulos de polímero en la célula, se detectó en aproximada-mente el 10% de los aislados heterotrofos replicados. La cinética de acumu-lación de PHA en Pseudomonas putida, P. oleovorans y Escherichia coli secomparó con la de los aislados ambientales por espectrofluorometría. Entodas las cepas estudiadas, la acumulación de PHA, medida como la intensi-

Crivado espectrofluorométrico rápido de bactérias produtoras de polihidroxialcanoatosem tapetes microbianos

Resumo. Os ecossistemas dos tapetes microbianos se caracterizam poroscilações diárias e estacionais em diversas variáveis físico-químicas, deforma tal que os microorganismos residentes devem adaptar-se frequente-mente às condições cambiantes de seu ambiente. Se destacou que em condi-ções de stress, as células bacterianas com conteúdos mais elevados em poli-hidroxialcanoatos (PHA) sobrevivem mais tempo que aquelas cujo conteúdoé menor. Neste estudo, utilizou-se a técnica do corante vermelho Nilo paraseleccionar as cepas produtoras de PHA des tapetes microbianos do delta doEbro e para monitorizar a acumulação relativa de PHA durante o cultivo nolaboratório. O número de isolados heterotrofos em TSA foi de aproximada-mente 107 unidades formadoras de colónias/g tapete microbiano. Destas,replicaram-se 100 colónias escolhidas ao acaso cultivadas em agar mineralsalino limitado em nitrogénio, ao que se lhe acrescentou o corante vermelhoNilo para a detecção de PHA. A fluorescência laranja, que se produz ao unir-se o vermelho Nilo aos grânulos de polímero na célula, detectou-se em apro-ximadamente 10% dos isolados heterotrofos replicados. A cinética de acu-mulação de PHA em Pseudomonas putida e P. oleovorans foram compara-das com os isolados ambientais por espectrofluorometría. A acumulação dePHA, medida como a intensidade relativa de fluorescência, alcançou uma

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dad relativa de fluorescencia, alcanzó una concentración estable a las 48 hde incubación. A las 72 h, la máxima intensidad de fluorescencia de las cepasincubadas con glucosa o fructosa fue casi siempre similar. La cepa MAT-28acumuló más PHA que los otros aislados. Estos resultados ponen de mani-fiesto que los datos obtenidos a partir de aislados ambientales pueden sermucho mejores que los que se basan en programas de simulación, y que lostapetes microbianos constituyen una excelente fuente de cepas productorasde PHA con aplicaciones industriales. [Int Microbiol 2006; 9(2):95-102]

Palabras clave: Pseudomonas oleovorans · Pseudomonas putida ·Escherichia coli · poli-hidroxialcanoatos · tapetes microbianos · rojo Nilo ·espectrofluorometría · cribado (“screening”)

concentração estável em todas as cepas estudadas às 48 h de incubação. Às72 h a intensidade máxima de fluorescência de cada cepa incubada com gli-cose e frutose foi quase sempre similar. A cepa MAT-28 acumulou mais PHAque os outros isolados. Os resultados obtidos põem de manifesto que osdados dos isolados ambientais podem melhorar notavelmente os estudosbaseados em programas de simulação, e que os tapetes microbianos consti-tuem uma excelente fonte de cepas produtoras de PHA com aplicaçõesindustriais. [Int Microbiol 2006; 9(2):95-102]

Palavras chave: Pseudomonas oleovorans · Pseudomonas putida ·Escherichia coli · poli-hidroxialcanoatos · tapetes microbianos · vermelhoNilo · espectrofluorometria · crivado (“screening”)